Ab27313

Protein extracts were resolved by 1-DE under reducing conditions and electrotransferred to nitrocellulose membranes in semi-dry conditions (Trans-Blot Turbo system; BioRad, Hercules, CA, USA). Serum amyloid P (SAP) detection was performed using a mouse monoclonal antibody against total SAP (ab27313, 1:200 dilution, abcam, Cambridge, UK). Band detection was performed using a chemiluminiscent substrate dye (Luminata Forte Western HRP Substrate, Merck Millipore, Billerica, MA, USA) and a molecular imager ChemiDoc XRS System, Universal Hood II (BioRad, Hercules, CA, USA). Band quantification was performed with Image Lab 4.0 software (BioRad Laboratories, Hercules, CA, USA). Protein load was normalized with total protein staining, as previously described [23 (link)].

Stx2 was purified from the Stx2a-expressing RM10638 strain as previously described [24 (link)]. Stx1 was purchased from Toxin Technologies (Sarasota, FL, USA). Monoclonal antibodies against the B-subunits of Stx1, Stx1-1, and Stx1-2 were produced as previously described [25 (link)]. Monoclonal (Stx2-2) and polyclonal (Stx2-pAb) antibodies against Stx2 were prepared as previously described [26 (link),27 (link)]. Attachment of HRP to the Stx2-pAb and biotinylation of Stx1-2 was performed using a Lightning-Link HRP Conjugation Kit and Lightning-Link Biotin Conjugation Kit following the manufacturer’s instructions (Innova Biosciences, Cambridge, UK). HRP conjugated streptavidin was purchased from Invitrogen (Carlsbad, CA, USA). HuSAP was purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal antibody against HuSAP (ab27313) was purchased from Abcam (Cambridge, MA, USA).

Staining of Drosophila brain sections was carried out after ageing the flies for 25 days following eclosion. Sections (10 μm) were taken from the OCT blocks using a Microm HM 550 Cryostat (Microm International GmbH), then they were placed on Superfrost Plus slides (Menzel Gläser) and stored at -20°C until required for use. The sections were fixed in 4% w/v PFA for 10 min at room temperature (RT) and the slides were washed in PBS (3x 3min) followed by permeabilisation using 0.5% Tween-20 (in PBS) for 15 min at RT. This was followed by additional washing steps (PBS, 3x 3 min). The rest of the staining protocol was carried out as previously described in [15 (link)] when using ab36362 and ab45151 (Abcam). For staining with ab108508 and ab27313 (Abcam), the same protocol as described above was used and both antibodies were diluted 1:500.